Application of wheat gene TaPHR1 in improving salt tolerance of plants

Abstract

The invention discloses application of a wheat gene TaPHR1 in improvement of plant salt tolerance, and belongs to the technical field of functional genomics. Wherein the nucleotide sequence of the wheat gene TaPHR1 is as shown in SEQ ID NO: 2, the tolerance of a plant to salt stress can be improved through overexpression of the gene, and the plant is wheat or arabidopsis thaliana. The wheat gene TaPHR1 has the beneficial effects that the wheat gene TaPHR1 is transformed into a model plant arabidopsis thaliana, so that the salt tolerance of the arabidopsis thaliana can be obviously improved, if the wheat gene TaPHR1 is transformed into plants such as wheat, rice, corn and cauliflower, new germplasm with salt tolerance can be possibly obtained, and the new germplasm can be used as a new germplasm. The method is of great significance to cultivation of new varieties of excellent crops and wide application of the new varieties of the excellent crops in production.

Description

Technical Field

[0001] This invention relates to the application of a gene, specifically the application of the wheat gene TaPHR1 in improving plant salt tolerance, and belongs to the field of functional genomics technology. Background Technology

[0002] Salt stress is a severely adverse environmental factor that causes osmotic stress, cellular ion toxicity, and oxidative stress in crops, leading to inhibited seed germination, reduced photosynthetic performance, and stunted growth, significantly impacting crop yield. Soil salinization has become one of the major adverse factors affecting wheat growth and yield. Therefore, breeding salt-tolerant varieties is an effective measure to improve wheat salt tolerance, and further identifying and discovering key salt-tolerant genes is a scientific and rational biotechnological approach for breeding salt-tolerant varieties. Summary of the Invention

[0003] This invention screened and cloned the wheat gene TaPHR1 from Chinese spring (CS) wheat and confirmed that the gene can effectively improve the salt tolerance of Arabidopsis thaliana.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: Application of wheat gene TaPHR1 in improving plant salt tolerance, wherein the nucleotide sequence of wheat gene TaPHR1 is shown in SEQ ID NO: 2, and overexpression of this gene can improve the plant's tolerance to salt stress, wherein the plant is wheat or Arabidopsis thaliana.

[0005] Preferably, the aforementioned application includes the following steps: (1) PCR amplification and cloning of the wheat gene TaPHR1; (2) The cloned wheat gene TaPHR1 was ligated into an expression vector to obtain an overexpression recombinant vector; (3) The overexpression recombinant vector was transformed into Agrobacterium to obtain the overexpression recombinant strain; (4) Transform the overexpressing recombinant strain into plants, screen and obtain wheat gene TaPHR1 overexpressing lines.

[0006] More preferably, the aforementioned expression vector is super1300 (GFP-C); the aforementioned Agrobacterium is Agrobacterium tumefaciens GV3101.

[0007] The advantages of this invention are that transforming the wheat gene TaPHR1 into the model plant Arabidopsis thaliana can significantly improve the salt tolerance of Arabidopsis thaliana. If the wheat gene TaPHR1 is transformed into wheat, rice, corn, cauliflower and other plants, it is possible to obtain new germplasm with salt tolerance characteristics, which is of great significance for the breeding of superior new crop varieties and the widespread application of superior new crop varieties in production. Attached Figure Description

[0008] Figure 1 This is a schematic diagram of the structure of the target vector super1300 (GFP-C); Figure 2 These are PCR identification images of TaPHR1 overexpressing transgenic Arabidopsis lines (OE1, OE2, OE3); Figure 3 This is a statistical graph showing the relative expression levels of the TaPHR1 gene in the TaPHR1-overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) and the wild-type (WT) Arabidopsis thaliana ecotype in Colombia. Figure 4 This is a statistical chart showing the germination rate of TaPHR1 overexpressing transgenic Arabidopsis seeds (OE1, OE2, OE3) and wild-type Arabidopsis seeds (WT) of the Colombian ecotype Arabidopsis under normal conditions and after salt stress treatment. Figure 5 This is a comparison chart of root growth of each strain under normal conditions (control group); Figure 6 This is a statistical chart of root lengths for each strain under normal conditions (control group); Figure 7 This is a comparison chart of root growth of different strains under salt stress treatment; Figure 8 This is a statistical chart of root lengths for each strain under salt stress treatment. Figure 9 This is a comparison chart of plant heights of different lines under normal conditions and under salt stress conditions; Figure 10 This is a statistical chart of plant height for each line under normal conditions and under salt stress treatment. Figure 11 This is a comparison chart of the growth of each strain under normal conditions and under salt stress treatment. Figure 12 This is a statistical chart of the fresh weight of each strain under normal conditions and under salt stress treatment. Figure 13 This is a statistical chart of the dry weight of each strain under normal conditions and under salt stress conditions; Figure 14 This is a statistical chart of the relative conductivity of each strain under normal conditions and under salt stress conditions; Figure 15 This is a statistical chart of SOD activity of various strains under normal conditions and under salt stress conditions; Figure 16 This is a statistical chart of POD activity for each strain under normal conditions and under salt stress conditions; Figure 17This is a statistical chart showing the proline content of each strain under normal conditions and under salt stress conditions; Figure 18 This is a statistical chart of H2O2 content in each strain under normal conditions and under salt stress treatment. Figure 19 This is a statistical chart showing the MDA content of each strain under normal conditions and under salt stress treatment. Detailed Implementation

[0009] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Unless otherwise specified, the reagents, materials, and instruments used in the embodiments are commercially available. The terminology used in the embodiments generally has the meanings commonly understood by those skilled in the art, unless otherwise stated. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods.

[0010] Example 1: Cloning of the wheat gene TaPHR1 Wheat is an important food crop with a wide planting range, and it is also a crop with a strong ability to adapt to harsh external environments. This invention attempts to screen for salt-tolerant genes in wheat, and ultimately screened a salt-tolerant gene from Chinese spring (CS) wheat. In the Ensembl Plants database, the gene number of this gene is TraesCS6D02G097000.1. It is located on wheat chromosome 6D, with an ORF (open reading frame) of 1161 bp, an mRNA length of 1352 bp, and a nucleotide sequence as shown in SEQ ID NO: 1. Its encoded product has 386 amino acids, a molecular weight of 42631.63 g / mol, and an isoelectric point of 6.7343. This invention names this gene TaPHR1 and clones it using cloning technology. The specific steps are as follows: Total RNA was extracted from leaves of Chinese spring (CS) wheat grown hydroponically for 7 days using the Trizol method. After purification, cDNA was obtained by reverse transcription using HiScript III RT SuperMix for qPCR (+gDNA wiper). Using this cDNA as a template, the TaPHR1 gene was amplified using upstream primer L1 (SEQ ID NO: 3) and downstream primer R1 (SEQ ID NO: 4). The amplified product was sequenced to determine the nucleotide sequence of the target gene.

[0011] The nucleotide sequences of upstream primer L1 and downstream primer R1 are shown below: Upstream primer L1: 5'-ATGAGCACACAGAGTGTAATTC-3' (SEQ ID NO: 3); Downstream primer R1: 5'-TTCGGTGTCTGAAATTTTACGC-3' (SEQ ID NO: 4).

[0012] The PCR reaction system for amplifying the TaPHR1 gene was as follows: 8 μL ddH2O, 12.5 μL 2×Phanta Max Master Mix (DyePlus), 1 μL upstream primer L1, 1 μL downstream primer R1, and 2.5 μL cDNA template.

[0013] The PCR reaction conditions for amplifying the TaPHR1 gene were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 20 s, repeated for 34 cycles; 72℃ extension for 5 min, and finally stored at 4℃.

[0014] The obtained PCR amplification products were electrophoresed on a 1% agarose gel, and fragments with a sequence length of approximately 1158 bp were recovered. Then, the fragments were subjected to electrophoresis for 5 minutes. TM The TA / Blunt-Zero Cloning Kit was ligated to the pCE2 TA / Blunt-Zero and transformed into E. coli DH5α competent cells. Positive clones were selected for sequencing.

[0015] Sequencing results showed that a DNA fragment with a sequence length of 1158 bp was inserted into the pCE2 TA / Blunt-Zero vector. The nucleotide sequence of this DNA fragment, as shown in SEQ ID NO: 2, is consistent with the nucleotide sequence of the gene with gene number TraesCS6D02G097000.1 in the Ensembl Plants database after removing the stop codon, and is identified as the ORF sequence of the wheat gene TaPHR1.

[0016] Example 2: Construction of recombinant expression vector 1. Amplification of the target gene XbaI restriction sites were added to the 5' ends of upstream primer L1 and downstream primer R1, respectively, to obtain upstream primer L2 and downstream primer R2. The nucleotide sequences of upstream primer L2 and downstream primer R2 are shown below: Upstream primer L2: 5'-atacaccaaatcgactctagaATGAGCACACAGAGTGTAATTCCTG-3' (SEQ ID NO: 5); Downstream primer R2: 5'-cataggtacccgggctctagaTTCGGTGTCTGAAATTTACGCT-3' (SEQ ID NO: 6).

[0017] Using the positive Escherichia coli culture containing the pCE2 TA / Blunt-Zero recombinant vector obtained in Example 1 as a template, PCR amplification was performed.

[0018] The PCR reaction system consisted of: 8 μL ddH2O, 12.5 μL 2×Phanta Max Master Mix (Dye Plus), 1 μL upstream primer L2, 1 μL downstream primer R2, and 2.5 μL template.

[0019] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, repeated for 34 cycles; 72℃ extension for 5 min, and finally stored at 4℃.

[0020] PCR products (target gene) with a sequence length of approximately 1158 bp were recovered for subsequent reactions.

[0021] 2. Carrier linearization Using QuickCut TM XbaI restriction endonuclease was used to linearize and digest the target vector super1300 (GFP-C). The specific structure of the target vector super1300 (GFP-C) is shown in [link to documentation]. Figure 1 A linearized cloning vector was obtained.

[0022] Enzyme digestion system: plasmid 1 μg, 10× QuickCut Green Buffer 2 μL, QuickCut TM Add 1 μL of XbaI and bring the total volume to 20 μL with ddH2O.

[0023] Reaction conditions: 37℃ incubator for 30 minutes.

[0024] 3. Ligation of the target gene and the cloning vector The target gene obtained in step 1 was ligated into the linearized cloning vector obtained in step 2 using the CloneExpress II One step Cloning Kit (Vayme) to obtain the recombinant expression vector super1300(GFP-C)-TaPHR1.

[0025] The ligation system is as follows: 2 μL of linearized super1300 (GFP-C), 2 μL of target gene, 2 μL of 5×CE II Buffer, 1 μL of Exnase II, and 3 μL of ddH2O.

[0026] Reaction conditions: Gently pipette to mix the connection system, briefly centrifuge to collect the liquid at the bottom of the tube, and react at 37°C for 30 min.

[0027] 4. Screening and identification of target gene clones The specific steps for screening and identifying target gene clones are as follows: (1) Take 10 μL of the recombinant reaction product obtained in step 3 and transform it into competent Escherichia coli DH5α cells. Incubate at 37°C upside down for 12 h (the culture medium is LB solid medium with 50 μg / mL kanamycin sulfate added). (2) Pick the single clones obtained in step (1) and incubate them at 37℃ and 220rpm for 3h (the culture medium is LB liquid medium with 50μg / mL kanamycin sulfate added). (3) Using the bacterial culture obtained in step (2) as a template, PCR identification was performed using upstream primer L2 and downstream primer R2; (4) The bacterial culture that was positive by PCR in step (3) was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0028] The sequencing results of the TaPHR1 construct vector are shown in SEQ ID NO: 7. The reference sequence of the TaPHR1 construct vector is shown in SEQ ID NO: 8. The sequencing results show that the present invention has obtained a recombinant expression vector containing the target gene (TaPHR1), namely the recombinant expression vector super1300(GFP-C)-TaPHR1, and the recombinant expression vector super1300(GFP-C)-TaPHR1 is a linearized vector super1300(GFP-C) with the TaPHR1 sequence added at the XbaI site.

[0029] Plasmids extracted from correctly sequenced bacterial cultures were stored at -20°C for subsequent Agrobacterium-mediated transformation experiments.

[0030] Example 3: Transformation of the wheat gene TaPHR1 into Arabidopsis thaliana 1. Construction of recombinant Agrobacterium The recombinant expression vector super1300(GFP-C)-TaPHR1 prepared in Example 2 was transformed into Agrobacterium tumefaciens GV3101 competent cells and screened at 28°C in LB solid medium containing 50 μg / mL kanamycin sulfate and 20 μg / mL rifampin.

[0031] Positive single clones were picked and cultured at 28℃ and 220rpm for 12h (the culture medium was LB liquid medium supplemented with 50μg / mL kanamycin sulfate and 20μg / mL rifampin).

[0032] Take 1 μL of bacterial culture and perform PCR identification using upstream primer L2 and downstream primer R2.

[0033] The bacterial culture that tested positive by PCR was a recombinant Agrobacterium tumefaciens containing the recombinant expression vector super1300(GFP-C)-TaPHR1, and this recombinant Agrobacterium tumefaciens was named GV3101 / super1300(GFP-C)-TaPHR1.

[0034] 2. Obtaining TaPHR1 overexpression transgenic Arabidopsis The specific method for obtaining TaPHR1 overexpressing transgenic Arabidopsis thaliana is as follows: (1) Plant preparation before infection: Wild-type seeds of Colombian ecotype Arabidopsis thaliana were vernalized at 4℃ for 72h, sown in MS solid medium, and cultured in a culture room at 22℃, 16h light / 8h dark, and 60%-70% humidity. When the plants grew to two true leaves, they were transplanted into planting pots containing mixed culture medium (nutrient soil and vermiculite mixed in a mass ratio of 3:1). After the plants flowered, the top of the main branch was cut off (to promote the development of lateral branches). Watered thoroughly the day before infection. (2) Activation of Agrobacterium: 1 mL of recombinant Agrobacterium tumefaciens GV3101 / super1300 (GFP-C)-TaPHR1 bacterial culture was poured into a pre-sterilized 250 mL Erlenmeyer flask in a clean bench. 150 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and 20 μg / mL rifampin was added. The mixture was incubated in a shaker at 28 °C for 16 h until OD was reached. 600 Once the concentration reaches 0.8, the bacterial culture is dispensed into three 50mL centrifuge tubes, centrifuged at 8000rpm for 10min, the supernatant is discarded, and the bacterial cells are retained. (3) Preparation of resuspension: Dissolve 2.5g of sucrose in 50mL of distilled water, and then add 10μL of Silwet-L77; (4) Add the resuspension solution to the activated Agrobacterium cells in batches, 10 mL each time, and measure the OD while adding. 600 Continue until the concentration reaches 0.8, at which point a resuspended bacterial solution is obtained; (5) Infecting Arabidopsis thaliana by dipping flowers: At 9:00 a.m. (when the flowers are in full bloom, which is conducive to infection), each time use the tip of a pipette to take 1 mL of the mixed resuspended bacterial solution and drop it onto the Arabidopsis thaliana inflorescence for infection. After all the inflorescences are infected, they are immediately treated in the dark for 1 day. Water is then applied during the light period. A second infection is carried out one week later. (6) Harvest seeds of infected Arabidopsis plants: Arabidopsis plants that have undergone two-stage infection treatment are cultured using conventional methods until they bear fruit, and mature T0 generation seeds are harvested. (7) Cultivation of T0 generation positive seedlings: Disinfect the T0 generation seeds, then evenly spread them on MS solid medium containing 100μL / 100mL hygromycin. Cultivate until Arabidopsis seedlings grow true leaves, and some seedlings have healthy true leaves and roots that have penetrated the medium for a significantly longer period (about 14 days). T0 generation positive seedlings are obtained. The T0 generation positive seedlings are transferred to planting pots containing mixed culture medium (nutrient soil and vermiculite are mixed in a mass ratio of 3:1) for cultivation. Mature T1 generation seeds are harvested from each plant. (8) Following the method described in step (7), the T1 generation seeds are cultured into T1 generation lines containing hygromycin resistance. If the ratio of positive seedlings to dead seedlings is approximately 3:1, it is a single-copy line. Then, the plants in the planting pots are cultured to fruiting using conventional methods, and the mature T2 generation seeds produced on each individual plant in the T1 generation line are harvested. (9) Ten T2 generation seeds were randomly selected and screened for hygromycin resistance in the same way. Those that no longer produced hygromycin resistance segregation were homozygous lines. Finally, three TaPHR1 overexpressing transgenic Arabidopsis T2 generation homozygous lines were obtained. (10) Harvest the seeds of the T2 generation homozygous line of TaPHR1 overexpressing transgenic Arabidopsis thaliana (i.e., the T3 generation, denoted as OE1, OE2, and OE3 respectively) for further phenotypic identification and analysis.

[0035] T0 generation represents the seeds produced by the transformation of the current generation and the plants that grow from them; T1 generation represents the seeds produced by self-pollination of the T0 generation and the plants that grow from them; T2 generation represents the seeds produced by self-pollination of the T1 generation and the plants that grow from them; T3 generation represents the seeds produced by self-pollination of the T2 generation and the plants that grow from them; lineage represents the population of seeds or plants produced by self-pollination of the same plant from the previous generation.

[0036] Example 4: PCR identification of TaPHR1 overexpressing transgenic Arabidopsis thaliana Leaves were taken from the three TaPHR1 overexpressing transgenic Arabidopsis T2 generation homozygous lines (OE1, OE2, OE3) obtained in Example 3. DNA was extracted from the leaves using the CTAB method and PCR amplification was performed using upstream primer L1 and downstream primer R1.

[0037] Electrophoresis results of PCR products are shown below Figure 2 PCR identification results showed that lines OE1, OE2, and OE3 were all transgenic Arabidopsis thaliana lines overexpressing TaPHR1.

[0038] Example 5: Real-time quantitative PCR detection of TaPHR1 overexpressing transgenic Arabidopsis thaliana The root systems of homozygous transgenic lines (OE1, OE2, OE3) of generation T3 and wild-type lines of Arabidopsis thaliana ecotype (denoted as line WT) were photographed. Total RNA was extracted using the Trizol method, purified, and then reverse transcribed using HiScriptⅢRTSuperMix for qPCR (+gDNA wiper) to obtain cDNA. Using the Arabidopsis thaliana AtActin gene as an internal reference, real-time quantitative PCR was performed on the wheat gene TaPHR1 using qRT-PCR primers.

[0039] The ABI QuantStudio3 real-time quantitative PCR instrument was used, along with ChamQ™ SYBR Color qPCR Master Mix reagents. The expression level of the Arabidopsis thaliana AtActin gene was used as an internal control. Results were analyzed using a threshold method to quantify the real-time quantitative PCR results. A fluorescence threshold was set, and the cycle number Ct was determined within that threshold. The C value was calculated based on the Ct value, with C=2. -△Ct , △Ct=Ct 目的基因 -Ct 内参基因 The average C value of the three replicates was calculated as the relative expression level of the target gene.

[0040] The reaction system consisted of: 5 μL of 2×ChamQ SYBR Color qPCR Master Mix, 0.2 μL of 50×ROX ReferenceDye II, 0.2 μL of upstream primer (10 μM), 0.2 μL of downstream primer (10 μM), 0.5 μL of first-strand cDNA solution diluted 3 times, and 3.9 μL of ddH2O.

[0041] The qRT-PCR amplification program uses a three-step method: (1) Pre-denaturation: react at 95℃ for 3 min; (2) Cyclic reaction: 95℃ for 15s, 59℃ for 20s, for a total of 40 cycles; (3) Dissolution curves: 95℃ reaction for 15s, 60℃ reaction for 60s, 95℃ reaction for 15s.

[0042] The primer sequences used for real-time quantitative PCR in this step are as follows: Actin-L: 5'-ATGAGCCAGTACGATCACAA-3' (SEQ ID NO: 9); Actin-R: 5'-TTAGAAGCAAATGTCCAGGG-3' (SEQ ID NO: 10); qRT-L: 5'-ATGGCGGAAACTCTACGG-3' (SEQ ID NO: 11); qRT-R: 5'-CGGTGTCTGAAATTTTACGC-3' (SEQ ID NO: 12).

[0043] The qRT-PCR amplification results are shown below. Figure 3 .Depend on Figure 3 It can be seen that the expression level of exogenous gene TaPHR1 in lines OE1, OE2 and OE3 is significantly higher than that in line WT, that is, the expression level of exogenous gene TaPHR1 in transgenic Arabidopsis plants is significantly higher than that in wild-type plants.

[0044] Example 6: Phenotypic identification of TaPHR1 overexpressing transgenic Arabidopsis plants 1. Phenotypic identification of germination rate during germination of TaPHR1-overexpressing transgenic Arabidopsis lines Salt stress treatment medium: Add 39.9g of 1 / 2MS solid dry powder to two Erlenmeyer flasks containing 1L of distilled water, then add 8.775g of NaCl and 11.7g of NaCl respectively, stir thoroughly, adjust the pH to 6.0, sterilize at 121℃ for 15min, and pour the medium into round petri dishes when it cools to 40-50℃ to prepare salt stress treatment medium with a NaCl concentration of 150mM (150mM NaCl group) and salt stress treatment medium with a NaCl concentration of 200mM (200mM NaCl group). The control group (CK group) did not add NaCl.

[0045] The TaPHR1 overexpression transgenic Arabidopsis seeds (OE1, OE2, OE3) obtained in Example 3 and the wild-type seeds (WT) of Colombian ecotype Arabidopsis were spread evenly on salt stress treatment medium containing 150 mM NaCl and 200 mM NaCl, respectively, and a control group (CK group) was set up. The germination of seeds in each group was observed, and the germination rate was statistically analyzed after 10 days.

[0046] The germination rates of TaPHR1-overexpressing transgenic Arabidopsis seeds (OE1, OE2, OE3) and wild-type seeds (WT) of the Colombian ecotype Arabidopsis thaliana under salt stress treatment are statistically shown in the table below. Figure 4 .

[0047] Depend on Figure 4It was found that under normal conditions (CK group), the germination rate of TaPHR1-overexpressing transgenic Arabidopsis seeds (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis seeds (WT). Under low salt stress (150 mM NaCl group), the germination rate of TaPHR1-overexpressing transgenic Arabidopsis seeds (OE1, OE2, OE3) was significantly higher than that of wild-type Arabidopsis seeds (WT) by 11.36%, 12.12%, and 13.26%, respectively. Under high salt stress (200 mM NaCl group), the germination rate of TaPHR1-overexpressing transgenic Arabidopsis seeds (OE1, OE2, OE3) was significantly higher than that of wild-type Arabidopsis seeds (WT) by 5.88%, 10.59%, and 5.10%, respectively.

[0048] The above results indicate that the transgenic lines exhibit higher tolerance to salt stress during seed germination.

[0049] 2. Phenotypic identification of TaPHR1 overexpressing transgenic Arabidopsis thaliana seedlings Square plate salt stress treatment medium: Add 39.9g 1 / 2MS solid dry powder and 8.775g NaCl to 1L distilled water, stir thoroughly, adjust the pH to 6.0, sterilize at 121℃ for 15min, and pour the plates (square plates) when the medium cools to 40-50℃ to prepare a square plate salt stress treatment medium with a NaCl concentration of 150mM (square plate salt stress group). The control group (CK group) does not contain NaCl.

[0050] TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) and wild-type Arabidopsis thaliana ecotype (WT) were cultured in an incubator under normal conditions for 5 days. Then, they were transferred to a square plate salt stress treatment medium supplemented with NaCl for vertical culture (OE1-NaCl group, OE2-NaCl group, OE3-NaCl group, WT-NaCl group). A control group (OE1-CK group, OE2-CK group, OE3-CK group, WT-CK group) was set up. After 10 days of culture, the root length of the plants was photographed and statistically analyzed.

[0051] Root growth of each strain in the control group is shown in the figure. Figure 5 Root length statistics are shown in Figure 6 .

[0052] Depend on Figure 5 and Figure 6 It can be seen that, under normal conditions, the root length of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) is not significantly different from that of the wild-type Arabidopsis thaliana line (WT).

[0053] The root growth of each line in the salt stress group is shown in the figure. Figure 7 Root length statistics are shown in Figure 8 .

[0054] Depend on Figure 7 and Figure 8 It can be seen that under salt stress, the root length of the TaPHR1 overexpressing transgenic Arabidopsis lines (OE1, OE2, OE3) was significantly increased by 8.36%, 15.07%, and 22.24% compared with the wild-type Arabidopsis line (WT), respectively.

[0055] 3. Phenotypic identification of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines at the adult stage TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) and wild-type Arabidopsis thaliana ecotype (WT) were cultured in round petri dishes under normal conditions for 7 days. Then, they were transferred to plastic pots containing a mixed culture medium (nutrient soil and vermiculite mixed at a mass ratio of 3:1) and sealed with plastic wrap for 5 days. After that, the plastic wrap was removed and the plants were cultured under normal conditions for 10 days. Then, they were subjected to NaCl (200 mM) stress treatment (NaCl group) and normal treatment (CK group). On the 12th day of NaCl stress treatment, the plants were photographed and the plant height was statistically analyzed.

[0056] The growth status of each group of plants is shown in the figure. Figure 9 The plant height statistics are shown in [link to statistics]. Figure 10 .

[0057] Depend on Figure 9 and Figure 10 It can be seen that under normal conditions, the plant height of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of the wild-type Arabidopsis thaliana line (WT); under salt stress conditions, the plant height of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 24.16%, 15.73%, and 16.85% respectively compared with the wild-type Arabidopsis thaliana line (WT).

[0058] TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) and wild-type Arabidopsis thaliana ecotype (WT) were cultured in round petri dishes under normal conditions for 7 days. They were then transferred to plastic pots containing a mixed culture medium (nutrient soil and vermiculite in a 3:1 mass ratio), sealed with plastic wrap, and cultured for 5 days. The film was then removed, and the plants were cultured under normal conditions for 10 days. Following this, they were subjected to NaCl (300 mM) stress treatment (NaCl group) and normal treatment (CK group). During this period, all flower stalks were removed, leaving only the rosette leaves. On day 12 of NaCl stress treatment, photographs were taken, and the fresh weight, dry weight, relative conductivity, SOD and POD activities, proline content, hydrogen peroxide content, and MDA content of each group were measured.

[0059] The growth status of each group of plants is shown in the figure. Figure 11 .Depend on Figure 11 It can be seen that under normal conditions, the growth of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); under salt stress conditions, the growth of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was better than that of wild-type Arabidopsis thaliana lines (WT).

[0060] The results of the fresh weight statistics of each group of plants are shown in the figure. Figure 12 .Depend on Figure 12 It can be seen that under normal conditions, the fresh weight of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); under salt stress conditions, the fresh weight of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 109.66%, 127.52%, and 124.52% respectively compared with that of wild-type Arabidopsis thaliana lines (WT).

[0061] The dry weight statistics of each group of plants are shown in the figure. Figure 13 .Depend on Figure 13 It can be seen that under normal conditions, the dry weight of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of the wild-type Arabidopsis thaliana line (WT); under salt stress conditions, the dry weight of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) showed an increasing trend compared with that of the wild-type Arabidopsis thaliana line (WT), significantly increasing by 59.56%, 57.44%, and 58.80%, respectively.

[0062] The results of the measurements of plant height, fresh weight, and dry weight indicate that the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines have higher tolerance to salt stress.

[0063] The statistical results of the relative electrical conductivity of each group of plants are shown in the figure. Figure 14 .Depend on Figure 14 It can be seen that under normal conditions, the relative electrical conductivity of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of the wild-type Arabidopsis thaliana line (WT); under salt stress, the relative electrical conductivity of the TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly lower than that of the wild-type Arabidopsis thaliana line (WT), decreasing by 48.40%, 48.29%, and 50.13%, respectively.

[0064] When subjected to salt stress, cells are damaged, leading to electrolyte leakage and impairing vital functions, manifested as an increase in relative conductivity. The results showed that under salt stress, the TaPHR1-overexpressing transgenic Arabidopsis thaliana lines suffered less cell damage and less electrolyte leakage than wild-type Arabidopsis thaliana lines, indicating that the former exhibited stronger salt tolerance.

[0065] The results of SOD activity assays for each group of plants are shown below. Figure 15 .Depend on Figure 15 It can be seen that under normal conditions, the SOD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the SOD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 67.82%, 94.47%, and 85.35% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0066] The results of POD activity assays for each group of plants are shown below. Figure 16 .Depend on Figure 16 It can be seen that under normal conditions, the POD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the POD activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 104.63%, 117.97%, and 129.38% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0067] SOD and POD are the main components of the enzymatic antioxidant system that scavenge reactive oxygen species (ROS). Under salt stress, the SOD and POD activities of TaPHR1 overexpressing transgenic Arabidopsis lines (OE1, OE2, OE3) were significantly higher than those of wild-type Arabidopsis line (WT), indicating that the former has a stronger ability to scavenge ROS and can effectively reduce membrane lipid peroxidation levels, thereby mitigating the harm of salt stress to plants.

[0068] The results of proline content determination in each group of plants are shown below. Figure 17 .Depend on Figure 17 It can be seen that under normal conditions, the proline content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the proline content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly increased by 120.08%, 85.92%, and 90.08% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0069] Proline is an important osmotic regulator. Under salt stress, the proline activity of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly higher than that of wild-type Arabidopsis thaliana line (WT). This indicates that the former can more effectively reduce cell osmotic potential, enhance cell water absorption capacity, maintain cell volume stability under salt stress, protect protein and cell membrane structure, and thus help the plant adapt to the salt stress environment.

[0070] The results of H2O2 content determination for each group of plants are shown below. Figure 18 .Depend on Figure 18 It can be seen that under normal conditions, the H2O2 content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the H2O2 content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly reduced by 56.71%, 60.88%, and 60.29% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0071] The results of MDA content determination for each group of plants are shown below. Figure 19 .Depend on Figure 19 It can be seen that under normal conditions, the MDA content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was not significantly different from that of wild-type Arabidopsis thaliana lines (WT); however, under salt stress conditions, the MDA content of TaPHR1 overexpressing transgenic Arabidopsis thaliana lines (OE1, OE2, OE3) was significantly reduced by 61.09%, 60.67%, and 50.04% respectively compared with wild-type Arabidopsis thaliana lines (WT).

[0072] H2O2 is a reactive oxygen species (ROS), and excessively high concentrations can cause oxidative damage to plant cells, affecting their normal growth and development. MDA is the final product of membrane lipid peroxidation; elevated levels of MDA primarily harm plants by damaging cell membrane systems, hindering photosynthesis, and reducing antioxidant capacity. Under salt stress, the H2O2 and MDA contents of TaPHR1-overexpressing transgenic Arabidopsis lines (OE1, OE2, OE3) were significantly lower than those of the wild-type Arabidopsis line (WT), indicating that the former enhanced the antioxidant capacity of Arabidopsis, mitigated oxidative stress damage, and thus reduced the harm of salt stress to the plants.

[0073] In summary, transforming the wheat gene TaPHR1 into the model plant Arabidopsis thaliana can significantly improve the salt tolerance of Arabidopsis thaliana.

[0074] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of the wheat gene TaPHR1 in improving plant salt tolerance, characterized in that, The nucleotide sequence of the wheat gene TaPHR1 is shown in SEQ ID NO:

2. Overexpression of this gene can improve the plant's tolerance to salt stress. The plant is wheat or Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The application includes the following steps: (1) PCR amplification and cloning of the wheat gene TaPHR1; (2) The cloned wheat gene TaPHR1 was ligated into an expression vector to obtain an overexpression recombinant vector; (3) The overexpression recombinant vector was transformed into Agrobacterium to obtain the overexpression recombinant strain; (4) Transform the overexpressing recombinant strain into plants, screen and obtain wheat gene TaPHR1 overexpressing lines.

3. The application according to claim 2, characterized in that, The expression vector is super1300 (GFP-C).

4. The application according to claim 2, characterized in that, The Agrobacterium is Agrobacterium tumefaciens GV3101.

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